The Intrathymic Pathogenesis of Myasthenia Graviset al., 1977), thymocytes (Fuchs et al., 1980), and...

The Intrathymic Pathogenesis of Myasthenia Gravis

ARNOLD I. LEVINSONa,*, DECHENG SONGa, GLEN GAULTONb and YI ZHENGa

aAllergy and Immunology Section, University of Pennsylvania School of Medicine, Room 1014 BRB II/III, 421 Curie Boulevard, Philadelphia, PA19104, USA; bDepartment of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Room 357 BRB II/III, 421 Curie

Boulevard, Philadelphia, PA 19104, USA

The thymus is considered to play an important role in the pathogenesis of Myasthenia gravis,an autoimmune disease characterized by antibody-mediated skeletal muscle weakness. However, itsrole is yet to be defined. The studies described herein summarize our efforts to determine howintrathymic expression of the neuromuscular type of acetylcholine (ACh) receptors is involved in theimmunopathogenesis of this autoimmune disease. We review the work characterizing the expression ofneuromuscular ACh receptors in the thymus and advance a new hypothesis that examines theintrathymic expression of this autoantigen in disease pathogenesis.

Keywords: Myasthenia gravis; Thymus; Acetylcholine receptor; Intrathymic expression

INTRODUCTION

Myasthenia gravis (MG) is a disease characterized by

weakness of striated muscles. The weakness is due to

impaired neuromuscular transmission resulting from a

reduction of the number of receptors for the neurotrans-

mitter, acetylcholine (ACh) at the postsynaptic myoneural

junction. This reduction is caused by the action of anti-

acetylcholine receptor (anti-AChR) antibodies, reviewed

in Levinson et al. (1987). MG is a prototypic autoimmune

disease; the immune effector mechanisms and autoanti-

genic target have been delineated. However, the events

leading to the abrogation of self-tolerance to the

neuromuscular type of AChR (nAChR) remain a mystery.

The thymus gland has long been considered to hold the

key to solving this mystery, although the nature of its

involvement remains to be elucidated.

Interest in a pathogenic role for the thymus in MG has

been fueled by pathologic, clinical and immunologic lines

of evidence as reviewed by Levinson and Wheatley

(1995). Briefly, thymus glands of 60–70% of MG patients

demonstrate the histological pattern of germinal center

hyperplasia whereas another 10% display cortical

epithelial cell thymomas. Thymectomy, particularly in

young patients (,40 years of age) with thymichyperplasia, is followed by clinical improvement and

remains a first-line therapeutic intervention. nAChR-

specific B and T cells have been recovered from MG

thymus specimens but not control thymus tissue. This

indicates that the autoimmune effector cells populate

the diseased thymus.

EXPRESSION OF nAChR IN THYMUS

A major feature of the thymus that very likely represents

an important pathogenic link to MG is the expression of

nAChRs on cells in this organ as reviewed in Levinson and

Wheatley (1995). The issue of thymic expression of AChR

has attracted considerable interest, in part, because of the

pivotal role that self-antigen expression in the thymus

plays in tailoring the T cell repertoire. Moreover, the

expression of nAChRS on thymic cells represents the first

description of the “promiscuous” intrathymic expression

of an organ-specific self-antigen that is the target of an

autoimmune attack in the periphery. The early reports

prompted some investigators to propose that the thymus

might actually serve as a site of sensitization for this

autoantigen (Wekerle et al., 1978). Taken at face value,

this idea might be viewed as being incongruent with the

cardinal immunologic construct, noted above, namely that

thymic self-proteins, particularly those expressed on

epithelial cells orchestrate the induction of self-tolerance

(Klein and Kyewski, 2000).

The nAChRs are expressed in two major forms as

reviewed in Levinson (2001a). The so-called mature or

junctional form is expressed on innervated muscles and

the immature or fetal form is expressed on non-innervated

tissue. At the mature (innervated) myoneural junction,

nAChRs are comprised of four subunits labeled a, b, dand 1. Two alpha subunits and one each of the othersubunits are assembled, like the whalebone in a corset, to

form an asymmetric hourglass channel spanning the

membrane. Two alternatively spliced alpha subunit

ISSN 1740-2522 print/ISSN 1740-2530 online q 2004 Taylor & Francis Ltd

DOI: 10.1080/17402520400001769

*Corresponding author. E-mail: [email protected]

Clinical & Developmental Immunology, September/December 2004 Vol. 11 (3/4), pp. 215–220

isoforms have been characterized, P3A2 and P3Aþ. Thelarger P3Aþ isoform, which includes an additionalsequence of 25 amino acids between exons 3 and 4, is

found only in humans and other primates. In fetal muscle,

as in adult denervated muscle or nonjunctional membrane,

a g subunit replaces the 1 subunit found in mature,innervated muscle endplates.

In the analysis of nAChRs in the thymus, many

investigators have focused on the nAChR alpha subunit

(nAChRa) since it is the source of the important pathogenicT and B cell epitopes for the pathogenic autoimmune

response in MG (Oshima et al., 1990; Zhang et al., 1990;

Conti-Fine et al., 1998; Fuji and Lindstrom, 1988).

Expression of this subunit was originally reported on a

variety of thymic cells including epithelial cells (Engel

et al., 1977), thymocytes (Fuchs et al., 1980), and myoid

cells (Kao and Drachman, 1977; Schluep et al., 1987).

Myoid cells, which share phenotypic properties with

skeletal muscle cells, were originally viewed as the

principal AChR-expressing cells in thymus (Schluep et al.,

1987). They are found in the medulla of both normal and

MG thymus.

In the past several years, there has been renewed interest

in thymic cells as a source of nAChR expression. Several

investigators, including ourselves, have taken a molecular

approach in characterizing thymic AChRs and identifying

cell populations expressing them. Using reverse-transcrip-

tion-PCR (RT-PCR) technology, we reported that mRNA

for the AChRa was expressed in normal mouse(Wheatley et al., 1992), normal human and MG thymus

(Wheatley et al., 1993; Zheng et al., 1999). We also

reported that AChRa mRNA was expressed on trans-formed murine thymic cortical and medullary epithelial

cell lines and thymic dendritic cell lines (Wheatley et al.,

1992). We found that mRNAs encoding both major

isoforms of the human AChRa, i.e. P3Aþ and P3A2, wereexpressed in normal and MG thymus and normal human

thymic epithelial cells (Wheatley et al., 1993; Zheng et al.,

1999). Sequencing of P3Aþ and P3A2 cDNA clonesrecovered from control and MG thymus indicated that

they share the same nucleotide sequence as their

respective counterparts at the myoneural junction. Thus,

unless there are posttranslational changes, the structure of

the AChRa proteins expressed in the thymus and theperiphery are likely to be identical. These results provided

a structural basis for proposing that an immune response

directed at thymic nAChRa may be responsible forinitiating or perpetuating disease. Berrih-Aknin sub-

sequently reported that AChRa protein as well as mRNAwas expressed on human thymic epithelial cells (Wakkach

et al., 1978).

However, there is still a controversy over the expression

of the other subunits on thymic cells and whether they are

expressed as components of intact receptors. Some of the

reported discrepancies may reflect differences in the ages

of the thymus donors and differences in the design of the

RT-PCRs. At this time, it appears as if 1 and b mRNAs areexpressed in most normal and MG thymus specimens with

variable expression of d and g subunits (Naveneethamet al., 2001; Bruno et al., 2004). Expression of the AChR

subunits appears to be concentrated in the thymic

medullary compartment.

To gain a better understanding of how intrathymic

expression of nAChRa might be linked to the developmentof disease, we addressed additional features of nAChRaexpression in the thymus. We observed that the smaller

P3A2 isoform is present in approximately a five-fold

excess in both MG and control thymic tissue and a 2.5-fold

excess in a non-transformed human thymic epithelial cell

(TEC) line relative to the larger P3Aþ isoform (Fig. 1)(Zheng et al., 1999). The greater expression of the P3A2

isoform in thymus does not parallel its expression in

healthy and MG muscle tissue where both isoforms show

equivalent expression (Beeson et al., 1990). These

observations suggest that the expression of mRNAs

encoding the P3A2 and P3Aþ isoforms is regulateddifferently in human thymus and muscle. Since the same

disproportionate expression of P3A2 was observed in

control and MG thymus, it appears that the differential

pattern of expression observed in thymus relative to muscle

is not a manifestation of thymic pathology in MG. Rather,

this pattern may reflect control processes that are unique to

these two distinct tissue compartments. Presently, it is not

known whether the disproportionate expression of P3A2

vs. P3Aþ isoforms has pathogenic significance.We also observed that expression of both P3Aþ and

P3A2 mRNAs are increased in MG thymus compared to

control thymus (Fig. 2) (Zheng et al., 1999). This finding

parallels that reported for skeletal muscle where AChR

mRNA expression was found to be greater in MG muscle

than in control muscle (Guyon et al., 1993). The finding of

increased AChRa mRNA expression in MG thymus mayrepresent an attempt to compensate for the destructive

action of locally secreted anti-AChR antibodies. It is also

possible that the increased AChR mRNA expression may

reflect the antecedent action of other local environmental

factors, e.g. cytokines.

FIGURE 1 Relative expression of AChRa P3A2 and P3Aþ isoforms inthymus and TEC. Compilation of data from 14 MG thymuses, 7 controlthymuses, and 4 separate TEC experiments. The signals for the P3A2 andP3Aþ bands on Southern blots were quantitated on a phosphorimager.The P3A2/P3Aþ ratios are shown. Expression of P3A2 exceeded that ofP3Aþ by a factor of 5.5 ^ 0.9 (mean ^ SEM) in Control thymus,4.7 ^ 0.05 in MG thymus, and 2.8 ^ 0.2 in TEC. (Copyright: ClinicalImmunology, 1:1999).

A.I. LEVINSON et al.216

REGULATION OF INTRATHYMIC nAChRaEXPRESSION

IL-1 and IL-6 production by epithelial cells is increased in

hyperplastic thymic tissue obtained from MG patients

compared to thymus from control subjects (Cohen-

Kaminsky et al., 1978; Emilie et al., 1991). Since

cytokines produced in vitro by thymic epithelial cell

(TEC) lines demonstrate autocrine function (Galy and

Spits, 1991), it seemed plausible that these cytokines or

perhaps others produced by cells in the thymus might

regulate TEC expression of AChR. We examined this

possibility by incubating a human non-transformed TEC

line with either IL-1b, IL-4, IL-6 and interferon-g(IFN-g). We found that neither IL-1, IL-4, nor IL-6altered the expression of AChRa mRNA by this cell line(Zheng et al., 1999). By contrast, IFN-g increasedexpression of the P3A2 and P3Aþ isoforms by factors of2.7 and 2.8, respectively. It is known that IFN-gup-regulates the expression of MHC class II antigens on

thymic epithelial cells (Berrih-Aknin et al., 1985; Galy

and Spits, 1991). This dual effect of IFN-g on AChRa andMHC antigens raises the possibility that this cytokine, and

perhaps others, may alter expression of thymic AChRain vivo in a manner that leads to the development or

perpetuation of MG. Before addressing this idea, it would

be helpful to briefly review the role of self-antigen

expression in thymus plays in the development of T cell

tolerance and consider how the thymus could serve as a

site of immune activation.

INTRATHYMIC EXPRESSION OF SELF

ANTIGEN AND THE DEVELOPMENT OFT CELL TOLERANCE

The thymus plays a fundamental role in the generation of

the peripheral T cell repertoire as reviewed in Klein and

Kyewski (2000), Sprent et al. (1988), Kisielow and

Boehmer (1990), Alam et al. (1996) and Anderson et al.

(1996). It is generally believed that thymocytes with low

affinity receptors for self are positively selected for export

to the peripheral lymphoid tissues where they comprise the

T cell repertoire that recognizes exogenous antigens

(Kisielow and Boehmer, 1990; Anderson et al., 1996).

By contrast, T cell tolerance to self is effected largely by the

process of central deletion/inactivation. Developing

thymocytes with high affinity receptors for self-peptide

are silenced by apoptosis or anergy. There is widespread

agreement that presentation of self-peptides by cortical

epithelial cells is necessary for positive selection

(Kisielow and Boehmer, 1990; Alam et al., 1996;

Anderson et al., 1996). Thymic medullary epithelial cells

and to a lesser extent, bone marrow-derived macrophages

and dendritic are considered to be the major APCs

involved in negative selection (Blackman et al., 1990;

Bonomo and Matzinger, 1993; Hugo et al., 1994;

Hoffmann et al., 1995; Klein and Kyewski, 2000).

However, central deletion is not complete even though a

broad array of self-peptides is “promiscuously” expressed

on medullary thymic epithelial cells (Klein and Kyewski,

2000). Self-reactive T cells escape from the thymus in

small numbers, perhaps due to the fact that limiting levels

of self-antigen limit the efficiency of tolerance induction

(Adelstein et al., 1991; Iwabuchi et al., 1992; Oehen et al.,

1994). However, such self-reactive T cells are silenced by

their anergic or ignorant status, i.e. they never encounter

self-antigen in the periphery in a manner that leads to

immune activation, or they are suppressed by regulatory

T cells (Shevach, 2000).

FIGURE 2 Semiquantitative RT-PCR: compilation of results fromthymus specimens of seven control subjects and fourteen MG patients.The signal intensity of the AChRa bands is normalized to the signalintensity of the standard by calculating the ratio of thymicAChRa/AChRa standard. The expression of P3A2 and P3Aþ isoformsin MG thymus is 2.5- and 2.8-fold greater, respectively, than that incontrol thymus. (Copyright: Clinical Immunology, 1:1999).

FIGURE 3 Semiquantitative RT-PCR: compilation of results from sixTEC experiments depicting the effect of IFN-g on expression of AChRaisoforms in TEC. To determine the effect of IFN-g on the expression ofAChRa P3A2 and P3Aþ isoforms, we compared the normalized signalintensities of the isoforms (ratio of thymic AChRa/AChRa standard)detected in untreated and IFN-g treated TEC9 cultures. Expression ofP3A2 mRNA was significantly greater in IFN-g treated (2.19 ^ 0.75,mean þ SEM) than in untreated cultures (0.89 ^ 0.36, p , 0:05;student’s t-test). Likewise, expression of P3Aþ mRNA wassignificantly greater in IFN-g treated (0.9 ^ 0.31) than in untreatedcultures (0.36 ^ 0.15, p , 0:05). (Copyright: Clinical Immunology,1:1999).

PATHOGENESIS OF MYASTHENIA GRAVIS 217

THE THYMUS AND T CELL TRAFFICKING

Based on the classic studies of Gowans, traffic of

lymphocytes is generally considered to be unidirectional,

i.e. out of the thymus into the blood and peripheral

lymphoid organs (Gowans and Knight, 1964). However,

small numbers of peripheral immunocompetent T cells

migrate to the thymus, entering via the medulla

(Naparstek et al., 1982,1993; Michie et al., 1988;

Hirokawa et al., 1989; Agus et al., 1991; Gossmann

et al., 1991; Westermann et al., 1991). Most of the thymic

immigrants are T cells activated in the peripheral immune

system although even resting T cells may gain access to

the thymus (Hirokawa et al., 1989; Agus et al., 1991;

Gossmann et al., 1991; Westermann et al., 1991). It is not

known if the rate or number of thymic immigrants is

increased by an inflammatory reaction in the thymus.

Furthermore, it is not known if self-reactive T cell

immigrants are activated when they encounter their

specific antigens in the thymus. Thymus T cell immigrants

specific for the lymphocytic choriomeningitis virus

(LCMV) clear infectious foci from the thymus (Gossmann

et al., 1991). This observation indicates that peripheral T

cells can be activated when they engage specific foreign

antigens in the thymus in an appropriate context. When

self-reactive T cells encounter their antigens in other

tissue compartments in the presence of requisite

co-stimulatory signals, they can be activated to express

their differentiation program, reviewed in Mondino et al.

(1996). One mechanism that leads to a milieu that

promotes the abrogation of tolerance peripherally is

infection. Local infection can lead to the upregulation of

MHC antigens and co-stimulatory molecules on cells that

express low levels of self-antigens and thereby lead to

activation of autoreactive T cells (Mondino et al., 1996).

A NEW MODEL OF THYMIC INFLAMMATIONAND ITS IMPACT ON “RETROGRADE” T CELL

MIGRATION

Delineation of the molecular events, particularly in the

thymus, that trigger MG has been hampered by the lack of

a model system. Thymic pathology is not a feature of

experimental models of MG in rodents. Although such

models have provided insight into the pathogenesis of

MG, they have not served to elucidate the role played by

the thymus (Christadoss et al., 2000). To address this issue

we have developed a model of inflammation targeted to

the thymic medulla, the site of thymic entry by peripheral

T cells (Levinson et al., 2001b). We generated molecular

variants of the well characterized thymotrophic Gross

murine leukemia virus (G-MLV), GD17, that had

previously been shown to exclusively infect medullary

thymic epithelium following their intrathymic injection in

naı̈ve mice. The variants were constructed to allow for

easy casetting of a broad array of genes of interest. The

thymo-tropic MLV vectors were created by ligating a

425 bp fragment containing the U3 region of GD-17 into

the LTR backbone of the well defined M-MLV vector

LXSH.

The vectors used in our studies are presented in

linear form in Fig. 4. The parental LXSH vector includes

50 M-MSV LTR, the psi packaging site and 50 gagregion, the hygromycin resistance gene under control of

the SV40 promoter, and the 30 LTR of M-MLV. For ourexperimental protocol, we modified this vector by

insertion of the Lac z gene (LBSHG). We utilized

LBSHG and LXSHG as our experimental and control

vectors, respectively. As was true for GD17, we found that

these vectors also target expression of encoded genes to

the thymic medullary epithelium.

Balb/c mice were immunized to b-galactosidase (b-gal)and then injected intrathymically (i.t.) with the b-galencoding vector LBSHG or the control vector LXSHG.

Hematoxylin and eosin stained sections of thymus

obtained four days after i.t. injection of LBSHG, but not

LXSHG, showed obliteration of the cortical/medullary

architecture with marked cellular expansion of the

medulla. To determine whether this local inflammatory

reaction non-specifically augmented the entry of peri-

pheral T cells into the thymus, b-gal immunized micewere injected i.v. with a population of CFSE-labeled

CD4þ T cells specific for an unrelated antigenfour days after i.t. injection of LBSHG or LXSHG. The

CD4þ T cells were derived from a transgenic mouse

FIGURE 4 Schematic diagram of MLV-based vectors. The vectors used in these studies are presented in linear form. The parental LXSH vector includesthe 50 M-MCV LTR, the psi packaging site and the 50 gag region, the hygromycin resistance gene under the control of the SV40 promoter, and the 30 LTR ofM-MLV. Vectors are modified by insertion of either GD-17 U3 or LacZ. (Copyright: Annals of the New York Academy of Sciences, 998:2003).


bearing a T cell receptor that recognized an influenza

hemagglutinin peptide. Animals that received LBSHG had

4.2-fold more CFSE-labeled CD4þ thymic immigrantsthan animals that received the control vector.

Using this model, we have begun to examine a new

hypothesis bearing on the intrathymic pathogenesis of MG.

(Fig. 5). The hypothesis posits that an inflammatory

reaction to an unrelated antigen within the medulla of the

thymus facilitates entry of peripheral AChRa-reactiveCD4þ T cells that escaped central deletion. These cellsenter the thymus in the medullary compartment where they

encounter AChRa expressed on antigen presenting cells.The concomitant intrathymic inflammatory reaction

creates a milieu that favors activation of these cells, i.e.

upregulation of MHC class II antigens, co-stimulatory

molecules on APCs, and perhaps upregulation of

AChR expression. Presentation of AChRa epitopes to theCD4þ thymic immigrants leads to their activation, help forlocally stimulated aAChR-reactive B cells, the productionof anti-AChR antibodies, and the development of MG. The

rationale for this hypothesis is outlined in Table I.

CONCLUSION

There is considerable circumstantial evidence that the

thymus plays a pivotal role in the pathogenesis of MG.

Nevertheless, the pathogenic link remains to be forged.

We are re-examining the hypothesis that AChR expressed

in the thymus drives the pathogenic autoimmune response.

We have established a model of intrathymic inflammation

that is localized to the thymic medulla and demonstrated

that such an inflammatory process promotes the

nonspecific entry of peripheral CD4þ T cells into thethymus. We are currently exploiting this model to

determine whether (1) AChR-reactive CD4þ T cellhoming to the thymus is also augmented by a concurrent

intrathymic inflammatory response to an unrelated antigen

and (2) AChR-reactive T cell immigrants undergo

activation following their engagement of autoantigen in

this inflammatory milieu, provide help for the production

of anti-AChR antibodies by immigrant autoreactive B

cells, and thereby promote the development of a

myasthenic syndrome.

Acknowledgements

Studies described in this report were supported by a grant

from the Muscular Dystrophy Association and National

Institutes of Health grant AI 50058. The authors thank

Cecelia Willitt for assistance in preparation of the

manuscript.

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nAChRa-reactive CD4+ T cells can be found in the blood of healthydonors as well as MG patients.

nAChRa-reactive T and B cells are recovered from MG thymus but not“control” thymus.

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Peripheral T cells traffic to thymus where they enter the medulla.

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The Intrathymic Pathogenesis of Myasthenia Graviset al., 1977), thymocytes (Fuchs et al., 1980), and...

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